Cim plates 16

Invasion of rat Schwann cells (2x10⁵) through a 1:40 fibronectin matrix was carried out using the xCelligence system and CIM-Plates 16 (ACEA Biosciences) according to manufacturer’s instructions. Cells were cultured overnight in the presence or absence of 10ng/ml TGFβ prior to seeding and invasion was allowed to proceed for 48 hr. Measurements were taken at the point of maximal invasion, which corresponded to 4-6 hr after seeding.
For invasion of mouse Schwann cells, 5x10⁵ cells were placed in the upper well of Boyden Chamber (Millicel 8.0 μm pore size) in the presence or absence of TGFβ (10ng/ml). Invasion through fibronectin-coated (1:40) membrane was allowed to proceed for 48 hr before cells were fixed, stained with dapi (1:5000) and counted.

Proliferation and migration analyses were performed using a xCELLigence Real-Time Cell Analyzer (RTCA) (ACEA Biosciences, San Diego, CA, USA). For measurement of proliferation dynamics, cells were seeded in 200 µL of growth media at a density of 5000 cells/well into an E-plate 16 (ACEA Biosciences, USA). Proliferation curves were evaluated over the course of 72 h with measurements taken every 15 min. Doubling time was analyzed from 8 to 43 h using RTCA software 2.1.0 (ACEA Biosciences, USA). For measurement of migration dynamics, 15,000 cells/mL density were seeded in 100 µL of serum-free media in upper chambers of CIM-plates 16 (ACEA Biosciences). The lower chamber was filled with 160 µL of growth media as a chemoattractant. Cell migration was evaluated over the course of 24 h with measurements taken every of 15 min. Slopes of migration curves were analyzed from 15 to 23 h using RTCA software 2.1.0 (ACEA Biosciences, USA).